Autism is a brain disorder that begins in early childhood and persists throughout adulthood. It affects three crucial areas of development: communication, social interaction, and creative or imaginative play. Children with autism have difficulties in social interaction and communication and may show repetitive behaviors and have unusual attachments to objects or routines. Similar to other neuropsychiatric disorders, the mechanism(s) involved in autism development have yet to be fully determined. Studies have indicated brain development in autism is abnormally characterized by accelerated growth in early life that results in brain enlargement in childhood (Aylward et al., 2002; Courchesne et al., 2001; Tate et al. 2007). Accelerated brain growth is also a feature of acquired macrocephaly, a finding in many autistic children. Also along these lines, a positive association between increasing radiate white matter volume and motor skill impairment in children with autism has also been shown (Mostofsky et al. 2007). Although such results are not universal for all autistic patients, they do define a significant subgroup of affected patients (Acosta and Pearl 2004; Ben Bashat et al. 2007), which potentially could be spared disease if intervention could be made early in neurodevelopment.
Autism affects thousands of Americans and encompasses a number of subtypes, with various putative causes and few documented ameliorative treatments. The disorders of the autistic spectrum may be present at birth, or may have later onset, for example, at ages two or three. Autism has increased tenfold in the last fifteen years. It is estimated to afflict between 2 and 5 of every 1000 children and is four times more likely to strike boys than girls. It is estimated that about 1.5 million American children and adults have autism. The U.S. Department of Education has estimated the rate of autism is increasing at 10 to 17 percent each year.
Autism has a strong genetic component, and in some families, autism tends to be more prevalent. In identical twins with autism both are usually affected. However, the number of children with autism appears to be increasing more than expected for a genetic disorder. This suggests to scientists that genetic abnormalities require the influence of other factors to cause the disorder.
The main goals of treatment are to lessen associated deficits and family distress, and to increase quality of life and functional independence. No single treatment is best and treatment is typically tailored to the child's needs. Although there is no cure for autism, clinicians do agree that early detection could significantly improve chances in if not preventing it, then at last reducing its weakening effects. Current treatments were shown efficacious when applied earlier in child's life, prior to the full blown onset. Thus the early prognosis is a vital component in the fight against this disease.
IL-6 and JAK2/STAT3
Inflammation plays a key role in many neurological diseases as well as in neurodegenerative diseases displaying cognitive and behavioral impairments. Recent studies have shown that abnormal immune responses contribute to autistic pathogenesis in animals (Warren et al., 2005). Neuroglia inflammation has been observed in the brain tissue of autistic individuals from as young as 5 to as old as 45 (Honda, Michelle (2008) Flavonoids benefit oxidative stress and inflammation associated with autism, www.MichelleHonda.com) Studies have been conducted that show that autistic brains show evidence of inflammation in certain brain areas, most prominently in the cerebellum. Ongoing inflammation was also found in the fluid surrounding the brain and spinal cord (Vargas D. L., et al. (2004) Neuroglial activation and neuroinflammation in the brain of patients with autism, Annals of Neurology 57:67-81).
Sharp increases in IL-6 production have been shown under inflammatory conditions in the brain. The main sources of IL-6 are reactive astrocytes and microglia (Behrens, M. M. et al. (2008) Interleukin-6 mediates the increase in NADPH-oxidase in ketamine model of schizophrenia, The Journal of Neuroscience, 28(51):13957-13966). Interleukin 6 (IL-6) plays a key role in developing autistic phenotype in the mice (Smith et al., 2007). It is well known that IL-6 highly activates signal transducer and activator of transcription protein 3 (STAT3) signal pathway in vitro and in vivo (Ebong et al., 2004; Ogata et al., 1997). The binding of IL-6 to its receptor (IL-6R) generates a complex with the gp130 protein thus triggering activation of Janus-activated kinases (JAK). This is then critical for inducing phosphorylation of docking sites of the receptor for SHP2 (SH2 domain-containing tyrosine phosphatase) and STAT (signal transducers and activators of transcription) proteins (Heinrich et al., 1998). There are various STAT protein variants, which cause an array of signaling which can result in neuronal death (Giunta et al., 2006, 2007, & 2008) or growth/differentiation (Kamimura et al., 2003). Phosphorylated STAT3 molecules dimerize and then move to the nucleus, where they lead to transactivation of target genes largely associated with neuronal growth (Schindler & Darnell 1995).
JAK2/STAT3 pathways induced by IL-6 play a crucial role in cell proliferation, a phenomenon known to occur at excess levels in the brains of a significant proportion of autistic children. Various studies demonstrate IL-6 signaling through Stat3 modulates the growth and proliferation of both neurons directly or via inhibition of neuron death. In vitro, stimulated astrocytes and microglia produce IL-6 (Van Wagoner et al., 1999; John et al., 2003), and its chimeric derivatives rescue neurons preserve myelin basic protein production in hippocampal slices exposed to excitotoxic insult (Pizzi et al., 2004). IL-6 also supports the survival of cultured postnatal rat septal cholinergic neurons (Hamma et al., 1999). Moreover, IL-6 has been shown to be crucially involved in the development, differentiation, regeneration, and degeneration of neurons in the peripheral and central nervous system (Gadient & Otten, 1994 & 1997). Further, evidence to support IL-6/Stat3 mediated growth signaling was demonstrated in spinal cord-derived neural progenitor cells (NPCs) exposed to IL-6 and epidermal growth factor (EGF). The phosphorylation level of Jak2/Stat3 was determined and maximal phosphorylation occurred at 30 minutes of NPCs exposure to IL-6. In addition, phosphorylation of Jak2/Stat3 was attenuated by pre-treatment of cells with AG490, the JAK2 specific inhibitor, suggesting that this pathway play a key in repopulation and regeneration of spinal cord tissue after injury (Kang et al., 2008).
Maternal Infection Activation
The earliest point in neurodevelopment is fetal. During the neurodevelopment stage, it has been reported that activation/regulation of STAT3 signal pathways is critically involved (Yvonne et al. 2000). Maternal infection increases the risk of fetal neurological injury. Systemic inflammatory response during pregnancy may contribute to neuropsychiatric disease in childhood and adulthood (Huleihel et al., 2004). Epidemiological studies have shown that the risk of developing autism is associated with prenatal maternal infection. This presumably results from neurodevelopmental defects triggered by cytokine-related inflammatory events (Ashdown et al., 2006; Meyer et al., 2008), as there is increased incidence of autism in the offspring of mothers who suffered infections while pregnant (Patterson, 2005). Because of the strong genetic component in autism, it is likely that only genetically susceptible individuals who were exposed to maternal infection would develop the disorder. Thus, it has been suggested that the risk of autism associated with maternal infection may be considerably more than three to seven-fold in susceptible individuals.
Several studies suggest the maternal immune response or “activation” to infection, rather than infection of the fetus itself (Shi et al., 2003 & 2005), is responsible for the increased incidence of autism in the offspring of mothers who suffered infections while pregnant. After maternal immune activation (MIA) by influenza, LPS, or poly(I:C), cytokine levels are altered in the maternal serum as well as the amniotic fluid, placenta, and, most importantly, the fetal brain (Fidel et al., 1994; Cai et al., 2000; Urakubo et al., 2001; Gayle et al., 2004; Paintlia et al., 2004; Gilmore et al., 2005; Beloosesky et al., 2006; Meyer et al., 2006). Cytokines were initially characterized as compounds of the immune response, but have since been found to play a much broader, diverse role in physiology. Cytokines, including interleukin-6 (IL-6), IL-11, IL-27 and leukemia inhibitory factor, have been shown to confer signaling in both the developing and adult brain; both directly and indirectly modulating neuronal functions (Bauer et al., 2007). Therefore, it is not surprising that inflammatory cytokines and their receptors modulate brain morphology during development as well. Particularly, IL-6 is likely a central link between maternal infection and neurodevelopmental derangement (Akira et al., 1990; Smith et al., 2007).
Maternal MIA in pregnant rodents yields offspring with abnormalities in behavior, histology, and gene expression that mimic psychiatric features of autism. This suggests MIA as a useful model of the development of autism. Specifically, single maternal injection of IL-6 on day 12.5 of pregnancy caused prepulse inhibition (PPI) and latent inhibition (LI) deficits in adult offspring. Co-administration of anti-IL-6 in the poly (I:C) model of MIA prevented the PPI, LI, and exploratory and social deficits caused by poly(I:C) and normalized the associated changes in gene expression in the brains of the adult offspring. Most convincingly, MIA in IL-6 knock-out mice did not yield several of the behavioral changes observed in the offspring of wild-type mice after MIA.
MIA-induced cytokines confer both direct and indirect effects on the fetus. Two methods have been used to study these effects: injecting or up-regulating cytokines during pregnancy in the absence of MIA, or blocking endogenous cytokines or preventing their induction during MIA. A study of the role of TNF-α in LPS-induced fetal loss and growth restriction indicated injection of anti-TNF-α antibodies or an inhibitor of TNF-α synthesis [pentoxifylline] can reduce these effects of LPS. Conversely, injection of TNF-α alone can induce fetal loss (Siler et al., 1994 and Xu et al., 2006) which is significantly worse in IL-18 knockout mice, but not in IL-1α/β knockout mice (Wang et al., 2006).
Much of the previous investigations of cytokine mediation of MIA effects on neuropathology and behavior in the offspring have focused on IL-6. This cytokine is involved in the regulation of physiological processes including inflammation and neurodevelopment; making it a particularly appealing candidate molecule for MIA-induced neuropathology. Indeed, during neurodevelopment, the signal transducer and activator of transcription-3 (STAT3) pathway, activated by IL-6, maintains homeostasis between neuro- and gliogenesis (He et al., 2005 and Murphy et al., 2000).
In support, Samuelsson et al., 2006 intraperitoneally injected IL-6 in pregnant rats for 3 days resulting in severe effects on the offspring. An important finding was that IL-6 mRNA levels remain elevated in the hippocampi of the offspring at 4 and 24 weeks of age; indicative of the ongoing state of immune dysregulation in adult autistic brains. Spatial memory in the water maze, a hippocampal-dependent behavior, was observed in that study. Importantly, the IL-6-treated offspring displayed increased latency to escape and time spent near the pool wall. Therefore, prolonged exposure to elevated IL-6 in utero causes a deficit in working memory (for reviews see Patterson 2008).
Blocking endogenous IL-6 in MIA also supports the central role of this cytokine (Smith et al., 2007). Co-injection of anti-IL-6 antibody with maternal poly(I:C) blocks the effects of MIA on the behavior of the offspring. Further, maternal injection of poly(I:C) in an IL-6 knockout mouse results in normal behaving offspring. In addition, the anti-IL-6 antibody also blocks the changes in brain transcription induced by maternal poly(I:C) (Patterson 2008). Maternal injection of poly(I:C) induces expression of IL-6 mRNA in fetal brain and placenta, and this is also dependent on the IL-6 induced by maternal poly(I:C) (Patterson 2008) (E. Hsiao and P. H. Patterson, unpublished). Taken together these previous works by other groups indicate both direct and indirect (positive feedback loop) mechanisms for IL-6 mediated MIA in the context of aberrant fetal brain development which could lead to an autism-like phenotype (Patterson 2008).
Schizophrenia
IL-6 plays a role not only in the pathogenesis of autism but also in the pathogenesis of schizophrenia in the context of maternal immune activation. MIA is used to describe an increase in circulating maternal cytokines in response to an infection during pregnancy (Smith et al., 2007). The ensuing cytokine response and the highly susceptible developmental period in which it occurs may precipitate the neuropathological and behavioral deficits observed in autism and related disorders such as schizophrenia. Schizophrenia and autism can result from the interaction between a susceptibility genotype and environmental risk factors. IL-6 is critical for mediating the behavioral and transcriptional changes in offspring in which mothers were exposed to infection. There is evidence to indicate that the maternal immune response, as opposed to the direct infection of the fetus, is responsible for the increased incidence of schizophrenia and autism in the offspring of mothers who suffer infections during pregnancy (Smith et al., 2007).
Neuroprotection IL-6/STAT3 signaling against N-methyl-D-aspartate (NMDA)-induced neurotoxicity has also been demonstrated. Cultured cerebellar granule neurons (CGNs) from postnatal (eight-day) infant rats were chronically exposed to IL-6 for eight days, and then NMDA (100 mol/L) was administered to the cultured CGNs. By way of MTT, and TUNEL assays, as well as confocal laser scanning microscope (CLSM) and western blotting, it was shown that NMDA stimulation of cell death could be avoided by IL-6 treatment. The NMDA stimulation of the CGNs chronically pretreated with IL-6 caused a very large increase in neuronal vitality, as well suppression of neuronal apoptosis compared with that in the control neurons without IL-6 pretreatment. The levels of phospho-Stat3 are significantly higher in IL-6-pretreated CGNs than those in IL-6-untreated neurons. Ketamine is an NMDA receptor antagonist which produces psychosis in humans and exacerbates symptoms in schizophrenic patients. Ketamine has been shown to activate the innate immune enzyme NADPH-oxidase in the brain. IL-6 has been shown to be the downstream mediator of ketamine in the induction of Nox2 and to activate NADPH oxidase in the brain providing further evidence for the role of IL-6 in the pathogenesis of schizophrenia (Behrens et al. 2008).
Diabetes
There is also an interaction between IL-6 and insulin systems, specifically insulin-like growth factor-1 (IGF-1) (Venkatasubramanian, G. (2007) Pathogenesis of Schizophrenia & Autism: The Interaction between Interleukin and Insulin Systems, Journal of Neuroscience 27(40): 10695-10702). IGF-1 has a significant role in fetal development and has neuroprotective, anti-apoptotic properties that are crucial for the optimal development of the brain (Fowden, 2003; Dore et al., 1997). IL-6 inhibits the secretion and biological activity of IGF-1 (de Martino et al., 2000; Lazarus et al., 1993). Cerebral damage in fetal pro-inflammatory states is associated with high IL-6 and low IGF-1 levels (Hansen-Pupp et al., 2007). Elevated IL-6 and decreased IGF-1 levels are also shown in schizophrenia and autism (Potvin et al., 2007; Venkatasubramanian et al., 2007; Jyonouchi et al., 2001; Riikonen et al., 2006).
Patients with diabetes also have reduced serum levels of IGF-1 that occur in response to a state of insulin resistance. IGF-1 acts in concert with insulin and has an important role in maintaining glucose homeostasis and protein metabolism in type 1 diabetes (Simpson, H. L. et al. (2004) Insulin-like growth factor has a direct effect on glucose and protein metabolism, but no effect on lipid metabolism in type 1 diabetes, The Journal of Clinical Endocrinology & Metabolism 89(1):425-432). In addition, similarly to the levels shown in schizophrenia and autism, patients exhibiting insulin-resistant states such as in diabetes have been shown to have increased levels of IL-6 and decreased levels of IGF-1 (Glund, S. et al. (2007) Interleukin-6 directly increases glucose metabolism in resting human skeletal muscle, The Journal of the American Diabetes Association 56(6):1630-1637).
Flavonoids
Flavonoids, plant polyphenolic compounds abundant in fruits and vegetables, exhibit a wide variety of biological effects, including antioxidant free-radical scavenging and anti-inflammatory properties (Rice-Evans C, Packer L (1998) in Flavonoids in Health and Diseases, eds Rice-Evans C, Packer L (CRC, Boca Raton, Fla.), pp 329-395). The flavonoid luteolin (3′,4′,5,7-tetrahydroxyflavone), abundant in celery, green pepper, parsley, perilla leaf, and chamomile tea (Shimoi K, et al. (1998) Intestinal absorption of luteolin and luteolin 7-O-β-glucoside ire rats and humans. FIBS Lett 438:220-224), is of particular interest for modulating immune reactions as several studies comparing the anti-inflammatory properties of luteolin with other flavonoids such as quercetin, genistein, or hesperetin in peripheral macrophages found luteolin to be most potent (Xagorari A, et al. (2001) Luteolin inhibits an endotoxin-stimulated phosphorylation cascade and proinflammatory cytokine production in macrophages. J Pharmacol Exp Ther 296:181-187; Comalada. M, et al. (2006) Inhibition of proinflammatory markers in primary bone marrow-derived mouse macrophages by naturally occurring flavonoids: Analysis of the structure-activity relationship. Biochem Pharmacol 72:1010-1021).
Luteolin has been shown to inhibit LPS-induced NF-κB transcriptional activity in intestinal epithelial cells, mouse bone-marrow derived dendritic cells (Kim J S, Jobin C (2005) The flavonoid luteolin prevents lipopolysaccharide-induced NF-κB signaling and gene expression by blocking IκB kinase activity in intestinal epithelial cells and bone-marrow derived dendritic cells. Immunology 115:375-387), murine macrophage cells, and rat fibroblasts (Kim S H, et al (2003) Luteolin inhibits the nuclear factor-κB transcriptional activity in Rat-1 fibroblasts. Biochem. Pharmacol 66:955-963). In another study, luteolin inhibited LPS-stimulated TNF-α and IL-6 in a murine macrophage cell line. These studies suggest luteolin modulates cell signaling pathways activated by LPS and subsequent production of inflammatory cytokines.
Diosmin containing supplements have been in use in Europe for the past three decades. Diosmin is currently considered a vascular-protecting agent and has been used safely for treatment of chronic venous insufficiency/varicose veins, hemorrhoids, lymphedema, and diabetes (Le Lyseng-Williamson and Perry 2003; Mlakar and Kosorok 2005; Nicolaides 2005). Clinical trials have used doses of 500-2,000 mg per day orally for up to one year. Throughout these trials, diosmin demonstrated an excellent safety profile and were well tolerated. Adverse events with such complexes were rare; and when they occurred, they were always mild, and transient. The side effects typically observed were mild cases of digestive intolerance requiring no changes in treatment.
Pharmacokinetic studies demonstrated diosmin is rapidly transformed in the intestine to diosmetin, its aglycone form (Cova et al., 1992; Labrid 1994; Meyer 1994). Diosmetin is subsequently absorbed and distributed throughout the body with a plasma half-life of 26-43 hrs. Data from this study was used to help determine the shape of the dose response, and optimal dose for human clinical trials to test diosmin as an effective anti-Stat3 prenatal supplement in infected, or potentially infected pregnant mothers. Diosmin can be used as a prenatal supplement to defend against autism risk, just as folate is currently used as prophylaxis against neural tube defects in human offspring.
Park et al., 2008 found that EGCG inhibits STAT3 activation as an integral part of inhibition of keloid formation. In an earlier study it was found that silibinin, a flavonoid, inhibits constitutive activation of STAT3, and causes caspase activation and apoptotic death of human prostate carcinoma cells (Agarwal et al., 2007). Prior to this, it was demonstrated that in vivo treatment of SJL/J mice with quercetin, a flavonoid, (i.p. 50 or 100 μg every other day which is equal to 1.25 mg/kg/day or 2.5 mg/kg/day) ameliorates experimental autoimmune encephalitis (EAE) by inhibiting IL-12 production and neural antigen-specific Th1 differentiation. In vitro treatment of activated T cells with this same flavonoid quartering blocks IL-12-induced tyrosine phosphorylation of JAK2, TYK2, STAT3, and STAT4, yielding a reduced IL-12-induced T cell proliferation and Th1 differentiation (Muthian and Bright 2004).
Our studies indicated that IL-6 activates the JAK2/STAT3 pathway, as N2a neuronal cells and brain homogenates from newborn IL-6-induced MIA (IL-6/MIA) offspring showed increased neuronal JAK2/STAT3 phosphorylation. In adulthood, these mice showed deficits in social interaction, suggesting that not only does IL-6 activate the JAK2/STAT3 pathway, but that it is also involved in the abnormal behavioral pathologies observed in MIA offspring and autism and related disorders. Next we investigated if inhibition of JAK2/STAT3 signaling could attenuate MIA-induced pathologies. Previous research by our laboratory has shown that bioflavonoids such as epi-gallocatechin gallate (EGCG) or luteolin, inhibit IFN-γ induced STAT1 activation and attenuate production of pro-inflammatory cytokines in cultured and primary microglial cells (Giunta et al., 2006, Jagtap et al., 2009 and Rezai-Zadeh et al., 2008).
We investigated the prophylactic effects of two flavonoids which possess better bioavailability and safety than these previously tested compounds. These two flavonoids are, luteolin, and its structural analog, diosmin. We found that JAK2/STAT3 phosphorylation and signaling as well as behavioral abnormalities in IL-6 induced MIA offspring could be ameliorated with these naturally occurring compounds. Our results showed that administration of diosmin (10 mg/kg/day) was able to block the STAT3 signal pathway; significantly opposing IL-6-induced abnormal behavior and neuropathological abnormalities in MIA/adult offspring. Using guidelines put forth by the Food and Drug Administration (Reagan-Shaw et al., 2008), this 10 mg/kg/day dose in mice is equivalent to 0.81 mg/kg/day in humans which translates into 48.6 mg/day for a 60 kg person.
The present invention includes methods for the treatment of autoimmune disorders such as autism, schizophrenia, and diabetes through the administration of flavonoids. Flavonoids, luteolin, diosmin, and its aglycone form, diosmetin, were found to inhibit activation/phosphorylation of STAT3 induced by IL-6 in cultured neuronal cells. These flavonoids have a profound and dose-dependent effect on inhibiting STAT3 activation using PC12 and N2a cells as evidenced by markedly decreased STAT3 phosphorylation by Western Blot Analysis. This effect is mediated by a reduction in downstream productions of the STAT3 signal pathway. Pregnant mice (E4) were treated with diosmin in the presence or absence of mouse IL-6 and autistic phenotypes, including pathology and behavioral changes in these treated mice, and were compared with a control. It was found that diosmin attenuates IL-6-induced autistic phenotype in mice. Due to the nature of these compounds, they are also effective in treating other diseases including schizophrenia and diabetes.